GSM6813051: Biliary epithelial cells PC depleted, rep 1; Mus musculus; RNA-Seq
Mouse livers were perfused with saline, dissected down to approximately 2-4 mm3 portions and covered with a mix of 0.125 mg/ml Collagenase (Sigma-Aldrich) and Dispase (Life Technologies) in 2% FCS Advanced DMEM/F-12 (Thermo Fisher). This mix was incubated 1 hour at 37ºC and individual bile ducts were manually picked up, cleaned twice with 5%FCS DMEM/F-12 (Thermo Fisher) and transferred to Matrigel (Corning) for subsequent culture. Bile ducts were grown as three-dimensional organoids for 4 to 5 passages and then transferred to a two-dimensional culture in plates covered with 1mg/ml collagen (Sigma). 500ng of each total-RNA sample was used for RNA-seq library preparation, using the NEBNEXT Ultra II Directional RNA Library Prep Kit (NEB #7760) and the Poly-A mRNA magnetic isolation module (NEB #E7490) according to the manufacturer's protocol. AMPure XP beads (Beckman Coulter #A63881) were used to purify the cDNA library. Multiple indexing adapters were ligated to the ends of the ds cDNA to prepare them for hybridisation onto a flow cell, before 10 cycles of PCR were used to selectively enrich those DNA fragments that had adapter molecules on both ends and amplify the amount of DNA in the library suitable for sequencing. Following amplification, libraries were purified using AMPure XP beads. Completed libraries were quantified by fluorometry using the Qubit dsDNA HS assay and assessed for quality and fragment size using the Agilent Bioanalyser with the DNA HS Kit (#5067-4626).